Multi-annual survey of health status disturbance in the Bilbao estuary (Bay of Biscay) based on sediment chemistry and juvenile sole (Solea spp.) histopathology.

The Bilbao estuary (SE Bay of Biscay) is a recovering ecosystem whose sediments are still contaminated. They represent a potential risk for the biota including benthic and demersal species living in direct contact with the sediment. In this context, the present study aims to survey trends of the health status of the Bilbao estuary based on sediment chemistry and sole (Solea spp.) histopathology. Monitoring campaigns were carried out every autumn from 2011 to 2017 along the estuary. Contaminant levels were measured in sediments; liver, gills and gonads of juvenile fish were collected for histopathology. Overall, contaminant levels fluctuated throughout the years, with highest values recorded in the earlier years of the study period. Sole histopathology showed alterations of mild severity. Results permitted to assess the environmental health status of the Bilbao estuary during 7 years, although no clear temporal trend was detected. Longer-term monitoring programmes are necessary to confirm the ecosystem recovery.

Baseline levels of environmental genotoxicity and potential confounding factors using common sole (Solea solea) as sentinel organism.

Micronuclei (MN) and nuclear buds (NB) frequency was assessed monthly over a one-year period in erythrocytes of common soles (Solea solea) from the Basque continental shelf, a minor disturbed area, in order to determine baseline levels and potential biotic and abiotic confounding factors within biomonitoring purposes. Both genotoxic parameters presented seasonal variations, although only NB frequency was positively correlated with environmental variables (i.e. water temperature and salinity). On the contrary, MN and NB were not affected by age and gender. Therefore, samples of combined age and gender can be used for biomonitoring genotoxic effects, although sampling period together with water temperature and salinity should be thoroughly considered for NB. Overall, these findings are the first attempt to establish baseline MN (0.10-0.78‰) and NB (0.13-0.82‰) frequencies in common sole from Basque marine waters, which are useful for upcoming data comparisons and integration within genotoxicity evaluation procedures of similar marine environments under biomonitoring scopes.

Histopathological baseline levels and confounding factors in common sole (Solea solea) for marine environmental risk assessment.

Liver and gonad histopathology, biometric parameters and hepatic metal bioaccumulation were assessed monthly over a one-year period in common soles from the Basque continental shelf, in order to determine baseline levels and confounding factors within biomonitoring studies. Biometric parameters and hepatic metal bioaccumulation varied according to season and gender. Accordingly, hepatic histopathological traits presented seasonal variations related to the reproductive cycle. However, the hepatic histopathological index showed that seasonality and gender were not significant confounding factors. Conversely, the gonad histopathological index was modulated by season and gender. As for organ comparison, the liver endured more severe histopathological damage than the gonad. In brief, the sampling period and gender may not affect the estimation of hepatic histopathological indices for biomonitoring purposes. Nonetheless, due to different sensitivities to environmental 'noise' variables, the sampling period and gender differentiation should be thoroughly considered for the assessment of gonad histopathology, biometrics and metal bioaccumulation.

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components.

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR.

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv(BAS)) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv(BAS) increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea.

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34-38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.